In this study, we have demonstrated the viability of in vitro
selection for identifying deoxyribozymes that 3′-phosphorylate
DNA oligonucleotide substrates. Although the capture method
is not strongly selective for the intended 3′-phosphorylated
DNA terminus, one deoxyribozyme with 3′-kinase activity was
identified. For this new deoxyribozyme, 3′Kin1, the same
initially random (N40) region sequence can be used with many
different DNA substrate sequences by appropriate choice of the
fixed-sequence segment on the 3′-side of the initially randomregion. At the first nucleotide position of the fixed-sequence
segment, a T is favored, regardless of the DNA substrate
sequence. At the remaining positions of the fixed-sequence
segment, straightforward Watson−Crick base pairing should be
included. With this arrangement, almost any DNA substrate
that has sequence 5′-NKR-3′ can be 3′-phosphorylated, where
N denotes any oligonucleotide sequence, K = T or G, and R =
A or G. Certain other substrate sequences may also be 3′-
phosphorylated.